Tumor-free Mice Research Articles

Differential plasma cytokine variation following X-ray or proton brain irradiation using machine-learning approaches

PurposeX-ray and proton irradiation have been reported to induce distinct modifications in cytokine expression in vitro and in vivo, suggesting a dissimilar inflammatory response between X-rays and protons. We aimed to investigate the differences in cytokine profiles early following fractionated brain irradiation with X-rays or protons and their relationship with leukocyte subpopulations in rodents. Materials and methodsOur study utilized data from 80 tumor-free mice subjected to X-ray or proton brain irradiation in four fractions of 2.5Gy. Sixteen non-irradiated mice were used as the controls. Blood was collected 12h postirradiation to examine the profile of 13 cytokines. Correlation analysis, principal component analysis (PCA), and tree-based modeling were used to investigate the relationship between cytokine levels and leukocyte subpopulation variations following irradiation in the blood. ResultsRegardless of the irradiation type, brain irradiation resulted in a notable elevation in the plasma levels of IFN-γ and MCP-1. The use of either X-ray or proton beam had differential effect on plasma cytokine levels following brain irradiation. Specifically, X-ray irradiation was associated with significantly increased plasma levels of IFN-β, IL-12p70, and IL-23, along with a decreased level of IL-1α, in comparison to proton irradiation. Correlation analysis revealed distinct cytokine regulatory patterns between X-ray and proton brain irradiation. PCA highlighted the association of MCP-1, IL-6, TNF-α, IL-17A, and IFN-γ with neutrophils, monocytes, and naïve T-cells following X-ray irradiation. TNF-α and IL-23 levels correlated with naïve CD4+-cells following proton irradiation. Tree-based models demonstrated that high TNF-α level resulted in an increase in naïve T-cells, neutrophils, and monocytes, whereas low IL-6 level was associated with decreases in these cell counts. ConclusionOur findings revealed distinct inflammatory responses induced by X-ray irradiation in contrast to proton brain irradiation, as demonstrated by the differential regulation of cytokines in the bloodstream. Moreover, the study highlighted the association between specific cytokine levels and various leukocyte subpopulations. Further investigation is essential to accurately determine the impact of proton and X-ray brain irradiation on the inflammatory response and the efficacy of radiotherapy treatment.

A multi-institutional study to investigate the sparing effect after whole brain electron FLASH in mice: Reproducibility and temporal evolution of functional, electrophysiological, and neurogenic endpoints

Background and PurposeUltra-high dose-rate radiotherapy (FLASH) has been shown to mitigate normal tissue toxicities associated with conventional dose rate radiotherapy (CONV) without compromising tumor killing in preclinical models. A prominent challenge in preclinical radiation research, including FLASH, is validating both the physical dosimetry and the biological effects across multiple institutions. Materials and MethodsWe previously demonstrated dosimetric reproducibility of two different electron FLASH devices at separate institutions using standardized phantoms and dosimeters. In this study, tumor-free adult female mice were given 10 Gy whole brain FLASH and CONV irradiation at both institutions and evaluated for the reproducibility and temporal evolution of multiple neurobiological endpoints. ResultsFLASH sparing of behavioral performance on novel object recognition (4 months post-irradiation) and of electrophysiologic long-term potentiation (LTP, 5 months post-irradiation) was reproduced between institutions. Differences between FLASH and CONV on the endpoints of hippocampal neurogenesis (Sox2, doublecortin), neuroinflammation (microglial activation), and electrophysiology (LTP) were not observed at early times (48 h to 2 weeks), but recovery of immature neurons by 3 weeks was greater with FLASH. ConclusionIn summary, we demonstrated reproducible FLASH sparing effects on the brain between two different beams at two different institutions with validated dosimetry. FLASH sparing effects on the endpoints evaluated manifested at later but not the earliest time points.

Immune checkpoint inhibitor treatment does not impair ovarian or endocrine function in a mouse model of triple negative breast cancer.

Representing 15-20% of all breast cancer cases, triple negative breast cancer (TNBC) is diagnosed more frequently in reproductive-age women and exhibits higher rates of disease metastasis and recurrence when compared with other subtypes. Few targeted treatments exist for TNBC, and many patients experience infertility and endocrine disruption as a result of frontline chemotherapy treatment. While they are a promising option for less toxic therapeutic approaches, little is known about the effects of immune checkpoint inhibitors on reproductive and endocrine function. Our findings in a syngeneic TNBC mouse model revealed that therapeutically relevant immunotherapies targeting PD-1, LAG-3, and TIM-3 had no effect on the quality and abundance of ovarian follicles, estrus cyclicity, or hormonal homeostasis. Similarly, in a tumor-free mouse model, we found that ovarian architecture, follicle abundance, estrus cyclicity, and ovulatory efficiency remain unchanged by PD-1 blockade. Taken together, our results suggest that immunotherapy may be a promising component of fertility-sparing therapeutic regimens for patients that wish to retain ovarian and endocrine function after cancer treatment.

Revealing the effect of X-ray or proton brain irradiation on systemic inflammation and leukocyte subpopulation interplay in rodents.

The absolute lymphocyte count (ALC), lymphocyte-to-monocyte ratio (LMR), and neutrophil-to-lymphocyte ratio (NLR) offer convenient means to assess systemic inflammation post-cancer treatment, which influences treatment outcomes. Understanding these biomarker variations and leukocyte subpopulation interplay is crucial for optimizing radiotherapy. Herein, leukocyte subpopulations (T-CD4+, T-CD8+, B-cells, NK-cells, neutrophils, monocytes) during and after brain irradiation (using X-rays or Protons) in tumor-free mice were used to compute ALC, LMR, and NLR, on which radiation parameter influence was assessed by principal component analysis (PCA). NLR kinetics were further examined using modeling. Leukocyte subpopulations interplays and their response to radiation parameters were examined using PCA and correlation analysis. Under X-rays, ALC and LMR decreased, with ALC recovered to baseline after irradiation, but not LMR. Both X-rays and protons increased the NLR during irradiation, recovering in protons but not X-rays. Both irradiation volume and dose rate had a pronounced effect on the NLR. Leukocyte subpopulation interplay was observed under X-rays and protons, normalizing in the proton group by day 28. Lymphopenia was observed in all lymphocyte subpopulations under X-ray irradiation but not protons. The recovery patterns varied among the subpopulations. Neutrophil counts increased during irradiation, with the recovery of protons, but not X-rays, by day 28. Interplays between NK-cells and myeloid subpopulations were evident under X-rays but not protons. Importantly, no interplay was detected between myeloid cells and T/B-cells, indicating that LMR and NLR variations were primarily due to independent responses to brain irradiation. A tumor-free experimental mouse model was used to study the effects of brain radiotherapy on systemic immunity. When administering fractionated irradiation with a total dose of 20 Gy using a vertical beam to either the whole brain or hemi-brain, proton irradiation had fewer adverse impacts on the immune system compared to X-rays in tumor-free rodents.

Long-term intermittent caloric restriction remodels the gut microbiota in mice genetically prone to breast cancer

ObjectivesGut microbiota dysbiosis is among the risk factors for breast cancer development, together with genetic background and dietary habits. However, caloric restriction has been shown to remodel the gut microbiota and slow tumor growth. Here, we investigated whether the gut microbiota mediates the preventive effects of long-term chronic or intermittent caloric restriction on breast cancer predisposition. Methods10-week-old transgenic breast cancer-prone mice were randomly assigned to dietary groups (ad libitum, chronic caloric restriction, and intermittent caloric restriction groups) and fed up to week 81. Stool samples were collected at weeks 10 (baseline), 17 (young), 49 (adult), and 81 (old). 16S rRNA gene sequencing was performed to identify the gut microbiota profile of the different groups. In order to investigate the breast cancer gut microbiota profile within genetically predisposed individuals regardless of diet, mammary tumor-bearing mice and mammary tumor-free but genetically prone mice were selected from the ad libitum group (n = 6). ResultsIntermittent caloric restriction increased the microbial diversity of adult mice and modified age-related compositional changes. A total of 13 genera were differentially abundant over time. Pathogenic Mycoplasma was enriched in the re-feeding period of the old intermittent caloric restriction group compared with baseline. Furthermore, mammary tumor-free mice showed shared gut microbiota characteristics with mammary tumor-bearing mice, suggesting an early link between genetic predisposition, gut microbiota, and breast cancer development. ConclusionsOur study revealed the role of gut microbes in the preventive effects of caloric restriction against breast cancer development, implying the significance of diet and microbiome interplay.

A bi-adjuvant nanovaccine amplifying STING activation for cancer immunotherapy

The insufficient immune activation and immunosuppressive microenvironment restrict the therapeutic efficacy of cancer vaccines. Herein, we exploited a bi-adjuvant nanovaccine to amplify the activation of the stimulator of interferon genes (STING) for potent and durable anti-tumor immunity. STING agonist (MnO2 nanoparticles), toll-like receptor 9 agonist (CpG), and antigenic proteins or peptides were strategically formulated into nanovaccines (MnC NVs) by a one-step biomineralization method. The nanovaccine efficiently targeted dendritic cells of draining lymph nodes, in which CpG-mediated the stimulation of nuclear factor-kappa B pathway augmented STING signaling. RNA sequencing analysis validated the enhanced expression of relevant genes of type-I interferon signaling pathways. The priming of antigen-specific CD8+ T cells and humoral immune responses led to potent tumor growth inhibition and prolonged survival time in melanoma and human papilloma virus-derived tumor models. MnC NVs were able to synergize with immune checkpoint blockade therapy, with 80 % survival over 40 days in TC-1 tumor model. Moreover, rechallenging of tumor-free mice with homotypic cells resulted in complete tumor growth inhibition, suggesting the generation of a long-term memory effect. The simplified nanovaccine platform provides a safe and robust strategy to boost STING-mediated anti-tumor immunity for personalized cancer immunotherapy, showing significant potential for clinical translation.

Cardiotoxicity in tumor-bearing mice receving thoracic radiation plus tumor radiation and PD-1 inhibitor therapy.

e20509 Background: At present, most preclinical studies explore the cardiotoxicity caused by radiotherapy combined with Anti-PD1s in tumor-free mice. we assessed the overall cardiotoxicity of radiotherapy combined with Anti-PD1, the possibility that blocking HMGB1 does not affect the anti-tumor effect, and the possibility that radiotherapy combined with Anti-PD1 reduces heart damage in tumor-bearing mice. Methods: 25 tumor-bearing C57BL/6 mice on both hind limbs were treated with anti-PD1 or PBS or Anti-HMGB1 with or without thoracic irradiation 20gy and right hind limb tumor 8gy×3F irradiation. HE staining was used to observe the cardiac structure and morphological changes. Flow cytometry was used to observe the distribution of CD3+, CD4+, CD8+T cells in cardiac and peripheral blood. Cardiac function was examined by echocardiography. The expression of myocardial injury marker LDH in peripheral blood was detected by Elisa. Results: Five group was established:A = Control;B = anti-PD1,C = RT,D = RT+Anti-PD1,E = RT+Anti-PD1+Anti-HMGB1.HE showed the myocardial injury in A(without any treatment) was the most severe,and and B and D was slightly severe than C group, administration of Anti-HMGB1 partially attenuated myocardial injury in D. Elisa showed the LDH of A was highest, B were consistent with D in LDH expression, RT was lower. Cardiac function showed no difference in ejection fraction and fractional shortening in five groups. Peripheral blood flow cytometry showed the absolute counts of CD3+T-cell and cd8+T cells were greatest in B group, followed by A, C, D, and E groups. Absolute CD4+ T cell values were highest in the B. The results of myocardial tissue infiltrating lymphocytes showed that the absolute CD3+T-cell and cd8+T cells counts were biggest in the B group, followed by A, C, D, and Egroups. Absolute CD4+T cell counts were highest in B group. C, D had the best anti-tumor effect, and the use of Anti-HMGB1 partially attenuated the cardiac injury caused by D group. Conclusions: (1)Without any treatment, the cancer cachexia induced by tumor will cause serious damage to myocardial. (2) The overall cardiotoxicity of radiotherapy combined with Anti-PD1 group was only slightly increased compared to RT or anti-PD1 alone. Maybe radiotherapy killed part of the Anti-PD1-activated lymphocytes, only resulting in a slight increase in cardiac infiltrating lymphocytes. Anti-HMGB1 maintain the anti-tumor effect of RT+Anti-PD1 and partially attenuated the cardiac damage. [Table: see text]

Dysfunctional CD8 T cells in tumors are addicted to antigen

Abstract Chronic antigen stimulation, as during cancer or chronic infection, leads to T cell dysfunction. We studied the impact of chronic antigen stimulation on T cell receptor (TCR) signaling in CD8 T cells using a genetic liver cancer mouse model. Tumor-specific CD8 T cells (TST) initially acquired proximal TCR signaling defects, but with increased antigen exposure showed both proximal and distal signaling defects. Despite impaired TCR signaling, a stable population of TST was maintained throughout tumorigenesis. We tried to rescue dysfunctional TST by removing them from tumors but found that TST failed to survive in tumor-free mice; thus, T cells are addicted to antigen, though antigen impairs TCR signaling. We investigated the mechanism of antigen-dependent TST persistence and found that throughout tumorigenesis, a subset of terminally-differentiated (TCF1-) TST within liver tumors could be found in cell cycle. The proliferation-associated distal TCR signaling pathways MAPK and mTOR were impaired in TCF1- TST, and we hypothesize that antigen-dependent persistence is NFAT-dependent. Studies are underway to determine whether all TCF1- TST can enter cell cycle in a stochastic, NFAT-driven manner, or if only a specific subset of TCF1- TST proliferate. By understanding how T cell signaling is regulated in response to chronic antigen stimulation, we can design strategies to selectively modulate TCR signaling to maintain proliferation while preventing loss of effector function.

Rescue of cardiac dysfunction during chemotherapy in acute myeloid leukaemia by blocking IL-1α.

Patients with acute myeloid leukaemia (AML) suffer from severe myocardial injury during daunorubicin (DNR)-based chemotherapy and are at high risk of cardiac mortality. The crosstalk between tumour cells and cardiomyocytes might play an important role in chemotherapy-related cardiotoxicity, but this has yet to be demonstrated. This study aimed to identify its underlying mechanism and explore potential therapeutic targets. Cardiac tissues were harvested from an AML patient after DNR-based chemotherapy and were subjected to single-nucleus RNA sequencing. Cardiac metabolism and function were evaluated in AML mice after DNR treatment by using positron emission tomography, magnetic resonance imaging, and stable-isotope tracing metabolomics. Plasma cytokines were screened in AML mice after DNR treatment. Genetically modified mice and cell lines were used to validate the central role of the identified cytokine and explore its downstream effectors. In the AML patient, disruption of cardiac metabolic homeostasis was associated with heart dysfunction after DNR-based chemotherapy. In AML mice, cardiac fatty acid utilization was attenuated, resulting in cardiac dysfunction after DNR treatment, but these phenotypes were not observed in similarly treated tumour-free mice. Furthermore, tumour cell-derived interleukin (IL)-1α was identified as a primary factor leading to DNR-induced cardiac dysfunction and administration of an anti-IL-1α neutralizing antibody could improve cardiac functions in AML mice after DNR treatment. This study revealed that crosstalk between tumour cells and cardiomyocytes during chemotherapy could disturb cardiac energy metabolism and impair heart function. IL-1α neutralizing antibody treatment is a promising strategy for alleviating chemotherapy-induced cardiotoxicity in AML patients.

Chemotherapy activates inflammasomes to cause inflammation-associated bone loss.

Chemotherapy is a widely used treatment for a variety of solid and hematological malignancies. Despite its success in improving the survival rate of cancer patients, chemotherapy causes significant toxicity to multiple organs, including the skeleton, but the underlying mechanisms have yet to be elucidated. Using tumor-free mouse models, which are commonly used to assess direct off-target effects of anti-neoplastic therapies, we found that doxorubicin caused massive bone loss in wild-type mice, a phenotype associated with increased number of osteoclasts, leukopenia, elevated serum levels of danger-associated molecular patterns (DAMPs; e.g. cell-free DNA and ATP) and cytokines (e.g. IL-1β and IL-18). Accordingly, doxorubicin activated the absent in melanoma (AIM2) and NLR family pyrin domain containing 3 (NLRP3) inflammasomes in macrophages and neutrophils, causing inflammatory cell death pyroptosis and NETosis, which correlated with its leukopenic effects. Moreover, the effects of this chemotherapeutic agent on cytokine secretion, cell demise, and bone loss were attenuated to various extent in conditions of AIM2 and/or NLRP3 insufficiency. Thus, we found that inflammasomes are key players in bone loss caused by doxorubicin, a finding that may inspire the development of a tailored adjuvant therapy that preserves the quality of this tissue in patients treated with this class of drugs.

Chemotherapy activates inflammasomes to cause inflammation-associated bone loss

Chemotherapy is a widely used treatment for a variety of solid and hematological malignancies. Despite its success in improving the survival rate of cancer patients, chemotherapy causes significant toxicity to multiple organs, including the skeleton, but the underlying mechanisms have yet to be elucidated. Using tumor-free mouse models, which are commonly used to assess direct off-target effects of anti-neoplastic therapies, we found that doxorubicin caused massive bone loss in wild-type mice, a phenotype associated with increased number of osteoclasts, leukopenia, elevated serum levels of danger-associated molecular patterns (DAMPs; e.g. cell-free DNA and ATP) and cytokines (e.g. IL-1β and IL-18). Accordingly, doxorubicin activated the absent in melanoma (AIM2) and NLR family pyrin domain containing 3 (NLRP3) inflammasomes in macrophages and neutrophils, causing inflammatory cell death pyroptosis and NETosis, which correlated with its leukopenic effects. Moreover, the effects of this chemotherapeutic agent on cytokine secretion, cell demise, and bone loss were attenuated to various extent in conditions of AIM2 and/or NLRP3 insufficiency. Thus, we found that inflammasomes are key players in bone loss caused by doxorubicin, a finding that may inspire the development of a tailored adjuvant therapy that preserves the quality of this tissue in patients treated with this class of drugs.

Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1365.

Abstract 4107: Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy

Abstract Background: In the past decade, immunotherapy has transformed cancer treatment, with the development of immune-checkpoint inhibitors and adoptive cell therapies. A novel frontier is personalized vaccines targeting neoantigens unique to tumor cells. Vaccines based on neoantigens have several advantages. First, neoantigens are exclusively expressed by tumor cells and can, therefore, elicit truly tumor-specific T cell responses, thereby preventing ‘off-target’ damage to nonmalignant tissues. Second, neoantigens are de novo epitopes derived from somatic mutations, which presents the possibility to circumvent T cell central tolerance of self-epitopes and thus induce immune responses to tumors. One of the major challenges is the optimal neoantigen discovery/selection. Our study combines in silico HLA binding models with the 'MHC-PepSeq' platform to enhance neoantigen prediction, creating a personalized neoantigen peptide vaccine. We aim to demonstrate its efficacy in a mouse CT26 colon cancer model. Methods: Our method involved whole exome sequencing of tumor and normal DNA to identify neoantigens expressed in CT26 tumors, generating a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay (PepSeq assay), to determine those exhibiting the highest binding affinity with mice HLAs. Vaccines were prepared by dissolving these peptides in sterile PBS. Mice (BALB/C) were subcutaneously implanted with CT26 tumors, then immunized with the vaccine via IV injection in combination with a TLR7/8 agonist. Mice received a second vaccine dose on Day 14, and some groups were treated with an anti-PD-1 agent. Tumors and spleens from the mice were collected, processed into single-cell suspensions, and analyzed by single-cell sequencing, TCR analysis, IHC, and flow cytometry assays. Peripheral blood mononuclear cells (PBMCs) were collected and utilized in MHC tetramer assays. Results: Preliminary in vivo studies demonstrated significant tumor growth restrictions (TGI around 70%) and prolonged tumor-free survival in some mice. Our data showed the generation of neoantigen-specific T cells in vaccinated mice. Remarkably, vaccinated, tumor-free mice, when re-challenged with the same tumor cells, maintained their tumor-free status, suggesting the vaccine induced post-treatment immunological memory. Combining the vaccine with checkpoint inhibition resulted in better tumor growth restriction in mice (TGI around 93%). Conclusion: Overall, our data suggests that our personalized neoantigen vaccine platform has the potential to evoke robust and durable immune responses and, when paired with suitable complementary therapies and immune adjuvants, may serve as an effective cancer treatment. Citation Format: Tithi Ghosh Halder, Kate Gutowsky, Serina Ng, Trason Thode, Alexis Weston, Erin Kelley, Jorge Soria-Bustos, Jorge A. Giron, Taylor Bargenquast, Mohan Kaadige, Sanjana Tripuraneni, Brian Durbin, Shelby Rheinschmidt, Sydney Adamson, Michael Gordon, Justin Moser, John Altin, Raffaella Soldi, Sunil Sharma. Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4107.

Abstract 6578: N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity

Abstract Background. Neutrophil elastase (ELANE) - a neutrophil-derived serine protease - kills a wide range of cancer cells without harming non-cancer cells through a unique killing program involving histone H1 and the death domain of CD95. Although ELANE attenuates tumor growth and induces anti-tumor immunity following intratumoral delivery (Cui et al., Cell, 2021), systemic delivery is stymied by the presence of serine protease inhibitors in blood (eg. alpha-1-antitrypsin (A1AT) which rapidly inhibit its enzymatic activity essential for its anti-cancer function. To overcome this challenge, we developed N17465, an enzyme that resists serine protease inhibitors in blood, and tested its effects on tumor growth, both as a monotherapy and in combination with checkpoint inhibitors. Methods. Enzymatic properties: N17465 enzymatic activity was quantified in the +/- of A1AT or plasma in vitro, and in blood following intravenous (I.V) injection into tumor-bearing mice. Enzyme activity assays included a fluorogenic substrate AAPV-AMC and the C-terminal domain of CD95 (amino acids212-335). Anti-cancer properties: For in vitro studies, cells were treated with N17465 and viability was quantified by calcein-AM and immunogenic cell death (ICD) markers. Cancer/non-cancer cells included human/murine cell lines and primary cells from ovarian cancer (OvCa) patients. For in vivo studies, N17465 was injected I.V and effects on tumor growth, immunology and survival were assessed as monotherapy or in combination with a checkpoint inhibitor (anti-CTLA4) in pre-clinical models. Results. N17465 maintained enzymatic activity following incubation with A1AT or mouse/human plasma in vitro, and following I.V injection into CT26 tumor-bearing mice in vivo. Importantly, protecting N17465 from serine protease inhibitors did not interfere with its ability to cleave CD95 (therapeutic target) or selectively kill cancer cells in vitro. Indeed, N17465 induced ICD markers, killed a wide range of murine/human cancer cells and also well tolerated by non-cancer cells. Systemically delivered N17465 produced durable tumor regression in a murine CT26 colon cancer model resulting in ~50% tumor-free mice, induced a favorable immune profile, and synergized with anti-CTLA4. N17465 also exhibited efficacy across a range of xenograft models spanning human colon, prostate, and lung cancer, as well as OvCa patient-derived CDX models. Conclusions. Altogether, our data demonstrate that N17465 selectively kills cancer cells, produces durable responses in mice, induces favorable immunology and synergies with checkpoint inhibitors in pre-clinical models. Its ability to maintain enzymatic activity in blood following systemic delivery, selectively kill a wide range of cancer cells, and stimulate anti-tumor immunity warrants further investigation of this unique therapeutic modality across a wide range of solid tumors. Citation Format: Ravindra Gujar, Chang Cui, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Lev Becker. N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6578.

Abstract 4457: Skeletal muscle-infiltrating CD8 T cells exacerbate muscle atrophy in a syngeneic lung cancer cachexia mouse model

Abstract Cancer-associated cachexia (CAC) is a devastating syndrome of irreversible body weight loss and muscle wasting. CAC has been reported to often lead to poor outcomes of immune checkpoint inhibitors (ICIs), emerging as a standard treatment for various types of cancer. Previously, we have demonstrated that KRAS-mutant lung tumor tissues display an immunosuppressive microenvironment with decreased CD8 T cells and increased regulatory T cells (Tregs). In this orthotopic lung cancer mouse model, treatment with an anti-PD-L1 antibody, a representative ICI, reduced tumor burden, whereas accelerated loss of body weight and skeletal muscle mass. We thus assumed that CD8 T cells could facilitate skeletal muscle wasting in lung tumor-bearing mice, a key feature of CAC. First, we established a syngeneic CAC mouse model through orthotopic transplantation of murine lung cancer cells. At 7 weeks post-injection, these mice developed CAC, displaying over 20 % body weight loss, almost total loss of epididymal fats, and profound skeletal muscle wasting. In line with our assumption, CAC mice showed an increased number of CD8 T cells but a reduced number of Tregs in skeletal muscles, compared to tumor-free control mice. We then investigated whether skeletal muscle-infiltrating CD8 T cells directly induce muscle atrophy indeed. In CAC mice, CD8 neutralization led to restoration of muscle mass and downregulation of muscle atrophy markers, but was not able to significantly affect lung tumor burden. Moreover, direct co-culture with splenic CD8 T cells exacerbated C2C12 myotube atrophy in a conditioned medium from lung cancer cells, further supporting the involvement of CD8 T cells in CAC. To elucidate its underlying mechanism, RNA sequencing analysis was conducted in both skeletal muscles and lung tumors of CAC mice. Based on the differently expressed gene analysis, Cxcl13 was upregulated in skeletal muscles of CAC mice, while downregulated in lung tumors. It is well documented that CXCL13 can mediate recruitment of CD8 T cells. In this regard, CXCL13 might be a plausible regulator in CD8 T cell-induced skeletal muscle atrophy, which requires further investigation to fully understand the molecular mechanism. Citation Format: Se-Young Park, Bomi Kim, Myunggyo Lee, Haeseung Lee, Na-Young Song. Skeletal muscle-infiltrating CD8 T cells exacerbate muscle atrophy in a syngeneic lung cancer cachexia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4457.

Abstract 3720: Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs

Abstract Background: Anti-PD-1 antibodies have been approved for a variety of carcinomas, but their efficacy in gastrointestinal cancers is limited. CD8-positive T cells (CD8) of tumor-infiltrating lymphocytes (TILs) and resident memory T cells (TRMs) with long-term anti-tumor immune function have been reported to be associated with the therapeutic efficacy of anti-PD-1 antibodies. Among our oncolytic adenoviruses (OVs), OBP-301, in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes, is currently undergoing clinical trials. In the present study, we investigated the usefulness of OVs as immunostimulants and their potential efficacy in combination with anti-PD-1 antibodies. Methods: The cell lines used were the mouse colon cancer cell line CT26 and the mouse pancreatic cancer cell line PAN02. In vitro, the cytotoxic activity of the OVs and extracellular ATP in the culture supernatant were determined. OBP-502, an OBP-301 variant with the gene cassette of the RGD peptide, and anti-PD-1 antibody were used to examine the antitumor effect on subcutaneous tumors. OBP-702, another OBP-301 variant with the gene cassette of the wild-type p53 tumor suppressor gene, was used to examine the effect of long-term anti-tumor immunity. The effects on memory T-precursor cells (MPEC), CD8 and TRM were immunologically assessed in a rechallenge study. Results: OBP-502 and OBP-702 showed cytotoxic activity against CT26 and PAN02 cells in vitro, and significantly increased ATP in the culture supernatant. In vivo, the anti-PD-1 antibody/OBP-502 combination therapy significantly increased intratumor CD8 in CT26 and PAN02 subcutaneous tumors compared to each monotherapy. Complete response was achieved in 4 out of 12 mice by the combination therapy against CT26 tumors. When CT26 cells was re-seeded into tumor-free mice cured by the combination therapy, 2 out of 4 mice remained tumor-free. In the CT26 and PAN02 subcutaneous tumor model, OBP-702 increased CD44-positive CD62L-negative MPECs that differentiated into TRMs in the spleen, and this effect was blocked by ATP receptor inhibition. In CT26 and PAN02 bilateral subcutaneous tumor models, OBP-702 had a significant antitumor effect on both OBP-702-treated and -non-treated sides, and TIL analysis showed a significant increase in CD8 and TRM. In a rechallenge study, OBP-702-pretreatment showed significant growth inhibition of re-inoculated tumors. Conclusion: The OVs showed potent and long-term immune activation by inducing CD8 and TRM in tumors as well as direct anti-tumor effects, and synergistic effects with ICI. Citation Format: Daisuke Kadowaki, Shinji Kuroda, Masashi Hashimoto, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Satoru Kikuchi, Kazuhiro Noma, Hiroshi Tazawa, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3720.

Abstract 51: OPB-101: An optimized mesothelin-specific CAR T cell product expressing CD8-targeted IL-2/15 and engineered to resist T cell exhaustion

Abstract T cell therapies have had modest efficacy in solid tumors due to their failure to proliferate following infusion. We have designed a mesothelin (MSLN) CAR T product (OPB-101) which includes a novel promoter (OP1), an optimized CAR, a safety switch, and a CD8α-targeted IL-2/15 designed cytokine to promote T cell expansion and improve efficacy in solid tumors. Human anti-MSLN binders were optimized for the target using our OUTSPACERTM library. CAR evaluation was conducted using in vitro coculture assays and validated NSG tumor xenograft models. OUTSMARTTM IL-2/15 was designed to avoid regulatory T cell activation by ablating IL-2Rα binding and re-targeted to CD8α to enhance CD8+ T cell and NK stimulation. EGFRoptTM was engineered for improved sensitivity to EGFR-targeted immunotherapies. Using 2A polypeptides, all transgenes were expressed with an antigen-dependent, inducible “stim-on” promoter (OP1) to regulate CAR expression, cytokine production, and resistance to exhaustion. The efficacy of the OPB-101 construct was tested in stringent exhaustion assays. In vivo efficacy was assessed with mice engrafted with s.c. H1650 tumors that were treated by i.v. injection of mock, CAR only or OPB-101 T cells at doses ranging from 0.25 × 106 to 2 × 106 cells. Tumor volume and weight were measured for 80 days. T cell levels and phenotype were measured by peripheral blood (PB) draws and intratumoral analysis (IT; day 14). OPB-101 efficiently killed MSLN+ tumor cell lines in repeat challenge and spheroid assays, and inducibly produced the IL-2/15 cytokine. IL-2/15 expression did not result in antigen-independent CAR T cell proliferation but extended survival in conditions without exogenous cytokine support. In vivo efficacy studies showed tumor control at 2 × 106 cells in the CAR only condition and modest anti-tumor efficacy at the 1 × 106 dose. In contrast, OPB-101 demonstrated complete and durable tumor elimination at the lowest dose of 0.25 × 106 cells (OPB-101 versus CAR only at 1 × 106 cells, p < 0.0001 and p < 0.0005 for donor 1 and 2, respectively). OPB-101 efficacy was associated with robust expansion in the PB (>100-fold) within IT (>5-fold) compared to the CAR only condition. OPB-101 PB and IT T cells showed reduced levels of PD-1, TIGIT and CD39 compared to CAR only suggesting resistance to exhaustion. Finally, studies conducted in tumor-free mice showed that OPB-101 expansion in vivo was dependent on the presence of tumor. In summary, OPB-101 using a regulated promoter (OP1) to dynamically express an optimized MSLN CAR, EGFRoptTM, and a CD8-targeted IL-2/15 cytokine enhances anti-tumor efficacy against solid tumors by promoting CAR expansion and persistence while limiting T cell exhaustion. These technologies allow for complete elimination of solid tumors at a low treatment dose (i.e., <250K cells) and may pave the way to more effective CAR T cell therapies for solid tumors. Citation Format: Rupesh Amin, Howell Moffett, Kevin Haworth, Jerry Chen, Jason Yokoyama, Allan Wang, Leah Tait, Maria Steele, John Crowl, Thaddeus Davenport, Joseph DeSautelle, Jared Hammer, Willimark Obenza, Vanessa Montoya, Robin Kirkpatrick, Laura Baker, Tina Tan, Kristie Shirley, Bradley Hammerson, Robert Langan, David Clausen, Paul Sample, Brian Weitzner, Shujun Yuan, Marc Lajoie, Scott Boyken, Aaron Foster. OPB-101: An optimized mesothelin-specific CAR T cell product expressing CD8-targeted IL-2/15 and engineered to resist T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 51.

Activation of GPR81 by lactate drives tumour-induced cachexia

Cachexia affects 50–80% of patients with cancer and accounts for 20% of cancer-related death, but the underlying mechanism driving cachexia remains elusive. Here we show that circulating lactate levels positively correlate with the degree of body weight loss in male and female patients suffering from cancer cachexia, as well as in clinically relevant mouse models. Lactate infusion per se is sufficient to trigger a cachectic phenotype in tumour-free mice in a dose-dependent manner. Furthermore, we demonstrate that adipose-specific G-protein-coupled receptor (GPR)81 ablation, similarly to global GPR81 deficiency, ameliorates lactate-induced or tumour-induced adipose and muscle wasting in male mice, revealing adipose GPR81 as the major mediator of the catabolic effects of lactate. Mechanistically, lactate/GPR81-induced cachexia occurs independently of the well-established protein kinase A catabolic pathway, but it is mediated by a signalling cascade sequentially activating Gi–Gβγ–RhoA/ROCK1–p38. These findings highlight the therapeutic potential of targeting GPR81 for the treatment of this life-threatening complication of cancer.

Early effects of different brain radiotherapy modalities on circulating leucocyte subpopulations in rodents

Purposes Lymphopenia is extensively studied, but not circulating leucocyte subpopulations, which however have distinct roles in tumor tolerance. Proton therapy has been shown to have a lesser impact on the immune system than conventional X-ray radiotherapy through lower dose exposure to healthy tissues. We explored the differential effects of brain X-ray and proton irradiation on circulating leucocyte subpopulations. Materials and methods Leucocyte subpopulation counts from tumor-free mice were obtained 12 hours after 4 fractions of 2.5 Gy. The relationships between irradiation type (X-rays or protons), irradiated volume (whole-brain/hemi-brain) and dose rate (1 or 2 Gy/min) with circulating leucocyte subpopulations (T-CD4+, T-CD8+, B, and NK-cells, neutrophils, and monocytes) were investigated using linear regression and tree-based modeling approaches. Relationships between dose maps (brain, vessels, lymph nodes (LNs)) and leucocyte subpopulations were analyzed and applied to construct the blood dose model, assessing the hypothesis of a direct lymphocyte-killing effect in radiation-induced lymphopenia. Results Radiation-induced lymphopenia occurred after X-ray but not proton brain irradiation in lymphoid subpopulations (T-CD4+, T-CD8+, B, and NK-cells). There was an increase in neutrophil counts following protons but not X-rays. Monocytes remained unchanged under both X-rays and protons. Besides irradiation type, irradiated volume and dose rate had a significant impact on NK-cell, neutrophil and monocyte levels but not T-CD4+, T-CD8+, and B-cells. The dose to the blood had a heterogeneous impact on leucocyte subpopulations: neutrophil counts remained stable with increasing dose to the blood, while lymphocyte counts decreased with increasing dose (T-CD8+-cells > T-CD4+-cells > B-cells > NK-cells). Direct cell-killing effect of the dose to the blood mildly contributed to radiation-induced lymphopenia. LN exposure significantly contributed to lymphopenia and partially explained the distinct impact of irradiation type on circulating lymphocytes. Conclusions Leucocyte subpopulations reacted differently to X-ray or proton brain irradiation. This difference could be partly explained by LN exposure to radiation dose. Further researches and analyses on other biological processes and interactions between leucocyte subpopulations are ongoing. The various mechanisms underlying leucocyte subpopulation changes under different irradiation modalities may have implications for the choice of radiotherapy modalities and their combination with immunotherapy in brain cancer treatment.

Drug-Loaded Tumor-Derived Microparticles Elicit CD8+ T Cell-Mediated Anti-Tumor Response in Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) poses significant challenges due to limited effective treatments and high recurrence rates. Immunotherapy, a promising approach, faces obstacles in HCC patients due to T-cell exhaustion and immunosuppression within the tumor microenvironment. Using doxorubicin-loaded tumor-derived microparticles (Dox-TMPs), the mice with H22 ascites model and subcutaneous tumors model were treated. Following the treatment, mice were re-challenged with H22 cells to compare the therapeutic effects and recurrence among different groups of mice, alongside examining the changes in the proportions of immune cells within the tumor microenvironment. Furthermore, Dox-TMPs were combined with anti-PD-1 to further validate their anti-tumor efficacy. In vitro studies using various liver cancer cell lines were conducted to verify the tumor-killing effects of Dox-TMPs. Additionally, CD8+ T cells from the abdominal cavity of tumor-free mice were co-cultured with H22 cells to confirm their specific tumor-killing abilities. Dox-TMPs demonstrate effective anti-tumor effects both in vitro and in vivo. In vivo, their effectiveness primarily involves enhancing CD8+ T cell infiltration, alleviating T cell immunosuppression, and improving the immune microenvironment to combat tumors. When used in combination with anti-PD-1, their anti-tumor effects are further enhanced. Moreover, some mice treated with Dox-TMPs developed anti-tumor immunity, displaying a self-specific T-cell immune response upon re-challenged with tumor cells. This suggests that Dox-TMPs also have the potential to act as a long-term immune response against tumor recurrence, indicating their capability as a tumor vaccine. Dox-TMPs exhibit a dual role in liver cancer by regulating T cells within the tumor microenvironment, functioning both as an anti-tumor agent and a potential tumor vaccine.